In recently years, microarray technologies enable the evaluation of up to tens of thousands of molecular interactions simultaneously in a high-throughput manner. DNA microarray-based assays have been widely used, including the applications for gene expression analysis, genotyping for mutations, single nucleotide polymorphisms (SNPs), and short tandem repeats (STRs), with regard to drug discovery, disease diagnostics, and forensic purpose (Heller, Ann Rev Biomed Eng (2002) 4: 129-153; Stoughton, Ann Rev Biochem (2005) 74: 53-82; Hoheisel, Nat Rev Genet (2006) 7: 200-210). Pre-determined specific oligonucleotide probes immobilized on microarray can serve as a de-multiplexing tool to sort spatially the products from parallel reactions performed in solution (Zhu et al., Antimicrob Agents Chemother (2007) 51: 3707-3713), and even can be more general ones, i.e., the designed and optimized artificial tags or their complementary sequences employed in the universal microarray (Gerrey et al., J Mol Biol (1999) 292: 251-262; Li et al., Hum Mutat (2008) 29: 306-314). Combined with the multiplex PCR method, microarray-based assays for SNPs and gene mutations, such as deletions, insertions, and indels, thus can be carried out in routine genetic and diagnostic laboratories.
Meanwhile, protein and chemical microarrays have emerged as two important tools in the field of proteomics (Xu and Lam, J Biomed Biotechnol (2003) δ: 257-266). Specific proteins, antibodies, small molecule compounds, peptides, and carbohydrates can now be immobilized on solid surfaces to form microarrays, just like DNA microarrays. These arrays of molecules can then be probed with simple composition of molecules or complex analytes.
Interactions between the analytes and the immobilized array of molecules are evaluated with a number of different detection systems. Typically, commercial use of microarrays employs optical detection with fluorescent, chemiluminescent or enzyme labels, electrochemical detection with enzymes, ferrocene or other electroactive labels, as well as label-free detection based on surface plasmon resonance or microgravimetric techniques (Sassolas et al., Chem Rev (2008) 108: 109-139). To further simplify the assay protocol and reduce the reliance on related equipment, magnetic bead labeling was employed so that assay results could be photographed with a charge-coupled device (CCD) assisted camera or viewed under low magnification microscope (Guo et al., J Anal Sci (2007) 23: 1-4; Li et al., supra; Shlyapnikov et al., Anal Biochem (2010) 399: 125-131), and cross-reactive contacts or unspecific bonds even can be quickly eliminated by applying magnetic field or shear flow (Mulvaney et al., Anal Biochem (2009) 392: 139-144). The detection of microarray-hybridized DNA with magnetic beads thus opens a new way to routine hybridization assays which do not require precise measurements of DNA concentration in solution.
Luminophores improve the detection of target-probe binding in microarray-based assays because they exhibit variations in signal intensity or emission spectra resulting from the binding of target-probe molecular complex. Theoretically, luminophore-labeling can be integrated with magnetic beads, facilitating the process of microarray-based assays. Luminophore-labeled molecules can be coupled to magnetic beads, each of which assembles a large amount of lumiphores at the same time, yielding high intensity of luminescence. High sensitivity detection of molecular interaction thus becomes possible.
Moreover, it's still highly desirable to further improve both sensitivity and specificity of microarray-based assays, concerning with the detection of various SNPs and gene mutations, particularly in clinical settings. The main hindrance of achieving this is that, as hybridization of labeled nucleic acid targets with surface-immobilized oligonucleotide probes is the central event in the detection of nucleic acids on microarrays (Riccelli et al., Nucleic Acids Res (2001) 29: 996-1004), only one of the two strands of DNA products is available to hybridize with these probes while the other one competes with the probes for the targets, acting as a severe interfering factor. Therefore, single-stranded DNA (ssDNA) should be enriched, and considering simplicity and cost-effectiveness, asymmetric polymerase chain reaction (PCR) was recommended in our previous work after comparing several most popular methods, and a one-step asymmetric PCR without purification process was also developed successfully with its enhanced sensitivity and specificity satisfying our requirements (Gao et al., Anal Lett (2003) 33: 2849-2863; Zhu et al., supra; Li et al., supra).
For rare clinical samples and their extreme importance of accuracy in detection, the one-step asymmetric PCR-based assay is incapable to deal with, due to its low sensitivity. An alternative way we did not recommend in the previous work was to employ microspheres, preferably paramagnetic microspheres due to their easy handling and good biocompatibility, which can be further improved with the concern of sensitivity (Gao et al., supra). Through capturing double-stranded DNA fragments with microspheres and removing the unwanted strands by denaturation methods, the yielded ssDNA products were hybridized with microarrays. Theoretically, the purer and more abundance the ssDNA products can be made, the better sensitivity is expected to achieve. As the common symmetric PCR has its properties of much higher amplification efficiency and easier design of multiplexing compared with asymmetric PCR, the combination of symmetric PCR and ssDNAs prepared with this method is expected to meet the above requirement.